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STATUS OF DETECTION OF MINIMAL
RESIDUAL DISEASE (MRD) IN ACUTE
LYMPHOBLASTIC LEUKEMIAS
DEPT OF MOL ONCOLOGY
CANCER INSTITUTE (WIA)
ADYAR, CHENNAI - 600 020
INTRODUCTION
o ALL MOST COMMON PEDIATRIC MALIGNANCY REGISTERED AT CANCER INSTITUTE (WIA)
o 30-40% T-ALL- WESTERN STUDIES – 85% B-ALL AND 15% T-ALL
• MCP 841 –80-90 % ACHIEVE CR BUT 30-40% RELAPSE
o PERSISTENCE OF LOW NUMBERS OF RESIDUAL LEUKEMIC CELLS – NOT DETECTABLE BY CONVENTIONAL CYTOMORPHOLOGICAL METHODS (1-5%)
o NEED FOR A SPECIFIC AND SENSITIVE METHOD TO DETECT/DEFINE MOLECULAR REMISSION/ RELAPSE (MINIMAL RESIDUAL DISEASE)
DETECTION OF MINIMAL RESIDUAL DISEASE IN T-ALL
WHY DETECT MRD?
o HELP ANTEDATE RELAPSE.
o THERAPY STRATIFICATION
o RISK STRATIFY PATIENTS
o ASSESS RESPONSE TO TREATMENT
o INTRODUCTION OF NEWER FORMS OF
BIOLOGICAL THERAPY WHEN TUMOUR
LOAD IS LOW
o EVALUATION AS A PROGNOSTIC MARKER
SENSITIVITY OF THE TECHNIQUES IN DETECTION OF MRD
NO. TECHNIQUE SENSITIVITY
1. MORPHOLOGY 1- 5% 2. CELL CULTURE SYSTEM 10-1 – 10-3
3. CYTOGENETICS 10-1 – 10-3
FISH 10-3
DUAL COLOR / TRIPLE COLOR INTERPHASE FISH
10-4
4 IMMUNOPHENOTYPE BY FLOW CYTOMETERY 10-3
MULTIPLE PARAMETER FLOW CYTOMETERY
10-4 – 10-5
5 SOUTHERN BLOT 1 – 5% 6. PCR 10-3 – 10—4
RT – PCR 10-4 - 10-5
REAL TIME QPCR 10-6
MARKERS USED FOR MRD IN ALL
o PCR ANALYSIS OF CLONE SPECIFIC JUNCTIONAL REGIONS OF TCR AND GENE REARRANGEMENTS
o PCR ANALYSIS OF BREAKPOINT FUSION TRANSCRIPTS OF LEUKEMIA SPECIFIC CHROMOSOMAL ABERRATIONS (BCR-ABL, TEL-AML,E2A-PBX, MLL-AF4. TAL-1 DELETION)
o MULTI PARAMETER FLOW CYTOMETRY
o QUALITATIVE AND QUANTITATIVE
TCR AND GENE REARRANGEMNTS
V D J 1
V1-J 1
DIVERSITY OF TCR BY T CELL DIFFERENTIATION-CORTICAL THYMOCYTES--V-D-J RECOMBINATION
V J1
V 1-J1
T-ALL ARREST IN DIFFERENTIATION
CLONAL PROLIFERATION OF ARRESTED CELL
EACH CELL IN CLONE --IDENTICAL JUNCTIONAL SEQUENCE
JUNCTIONAL REGION JUNCTIONAL REGIONRearranged
Germline
TCR AND ARE GOOD MARKERS FOR MRD – PCR
o LIMITED GERMLINE AND COMBINATORIAL DIVERSITY OF TCR AND GENES BUT EXTENSIVE JUNCTIONAL REGION DIVERSITY (LEUKEMIA SPECIFIC DNA FINGERPRINT) - DIFFERENT IN EACH LYMPHOCYTE AND EACH LYMPHOID LEUKEMIA.
o DEVISE PATIENT SPECIFIC PRIMERS/PROBES(ASO) o SOMATIC MUTATIONS NOT REPORTED IN REARRANGED TCR GENES
o IN 95% OF T-ALL, REARRANGED TCR AND JUNCTIONAL REGIONS OR BOTH ARE USED AS TARGETS FOR MRD-PCR
PITFALLS IN THE USE OF JUNCTIONAL REGIONS AS MRD PCR TARGETS
o FALSE POSITIVE
o BACKGROUND AMPLIFICATION OF SIMILAR
REARRANGEMENTS IN POLYCLONAL REACTIVE T
LYMPHOCYTES / NORMAL LYMPHOCYTES
o HETERO DUPLEX ANALYSIS – SIMPLE, FAST
CHEAP, RELIABLE METHOD TO CONFIRM CLONALITY
DETECTION OF MRD
DETECTION OF MINIMAL RESIDUAL DISEASE
INSTITUTE EXPERIENCE
o GENOMIC DNA -NORMAL & LEUKEMIC CELLS
o QUANTITATION -DIAGNOSIS , REMISSION, NORMAL
o ( SPEC)
o PCR AMPLIFICATION OF ABL, TCR AND TCR AT
PRESENTATION
o HETERODUPLEX ANALYSIS—PAGE
o HD BAND CUT ,ELUTED ,PCR REAMPLIFIED AND
o SEQUENCED TO DESIGN ASO (ALLELE SPECIFIC OLIGO)
o 50 CASES OF T-ALL STUDIED AT PRESENTATION o PCR–CLONALITY CONFIRMED BY HD ANALYSIS
o 24 CASES WERE AVAILABLE FOR FOLLOW-UP STUDIES
o DURATION OF FOLLOW-UP FROM 6 TO 72 MONTHS
o V1-J11.3/2.3 62.5% o V1 - J1 64%
o 2 V CLONAL MARKERS 17.5%
o V-J1 AND V1-J11.3/2.3 46%
DETECTION OF MINIMAL RESIDUAL DISEASE IN T-ALL -PCR-HDA
MRD-PCR FOLLOW UP – REMISSION / RELAPSE
ALL PATIENTS WERE PCR +VE/HD +VE AT END OF INDUCTION THERAPY (3 MOS) -MCP 841 o 6 PATIENTS IN CR BUT REVEALED CONTINUOUS PCR +VE/ HD +VE RELAPSED AND DIED
o COMBINATION OF PCR PRODUCTS AT PRESENTATION AND RELAPSE - SAME HD PATTERN - IDENTICAL CLONALITY
o ALL PATIENTS IN LONG TERM CR WERE HD –VE IN 8-12 MONTH REMISSION SAMPLES AND CONTINUED TO BE PCR –VE/HD -VE
Leukemia Research 2002 Vol 26, 335-43
RESULTS - MRD IN T-ALL-MCP 841
LEUKEMIA rESEARCH
HETERODUPLEX ANALYSIS
homo
hetero
homo
QUANTITATION OF MRD
QUANTITATION OF MRD o DETECT AND ACCURATELY ASSESS THE VOLUME OF PERSISTENT SUB -CLINICAL DISEASE -LEVELS AND DYNAMICS OF MRD o DEFINE THE EXTENT OF REDUCTION IN TUMOR VOLUME REQUIRED TO PREVENT RELAPSE AND ENSURE LONG TERM DISEASE FREE SURVIVAL
COMPETITIVE PCR
LIMITING DILUTION
REALTIME PCR
LABORIOUS, MORE AMOUNT OF DNA , RISK OF CONTAMINATION
QUANTITATION OF MRD – REAL TIME Q-PCR
• AFFORDS BOTH AMPLIFICATION AND ACCURATE QUANTIFICATION DURING EXPONENTIAL PHASE OF INITIAL TARGETS - SHORT TIME
• FLOURESCENCE IS MONITORED AND THE CROSSING POINT/THRESHOLD CORRELATES TO AMOUNT OF INITIAL COPIES OF TARGET
• NO NEED FOR GELS, RADIOACTIVITY AND POST -PCR MANIPULATION
• DETERMINATION OF LARGE DYNAMIC RANGE OF STARTING TARGET MOLECULE DETERMINATION
REAL TIME PCR TECHNIQUES
o SYBER GREEN 1 -BINDS TO DOUBLE STRANDED DNA
o HYBRIDISATION PROBE – DONOR AND ACCEPTOR FLUROCHROMES-FRET
o HYDROLYSIS PROBE -TAQMAN PROBE- 5’-3’ NUCLEASE ACTIVITY OF TAQ POLYMERASE
3 5
R Q
Fl quenched Fl emitted
Real time PCR-Amplificaton plot and Standard curve -
REAL TIME PCR -QUANTITATION OF MRD
1 ASO- PCR - SPECIFICITY AND SENSITIVITY ( 1 IN 10-5 )
2 ASO-PCR NORMAL DNA-NON SPECIFIC AMPLIFICATION
3 STANDARD CURVE PCR WITH KNOWN INTERNAL CONTROL-(RNASE P)(50 ng--50 pg) QUANTITATE SAMPLES
4 STANDARD CURVE PCR WITH ASO-J1 –SERIAL DILUTION OF PRESENTATION LEUKEMIC DNA (50ng--
5pg) IN 500ng OF NORMAL DNA. 5 REMISSION SAMPLE QUANTITATED USING ABOVE STD
CURVE
PROGNOSTIC VALUE OF MRD IN ALLWHEN AND HOW OFTEN SHOULD MRD BE MONITORED
SINGLE TIME POINT ANALYSIS IS INADEQUATE
AT LEAST 2 SERIAL MEASUREMENTS ARE NEEDED DURING EARLY MONTHS OF TREATMENT
1 AT END OF INDUCTION 1-RESPONSE TO TREATMENT
2 AT START OF CONSOLIDATION-RISK OF RELAPSE IS PROPORTIONAL TO MRD LEVELS-POWERFUL PROG NOSTIC MARKER
LOW RISK 10-3 INTERMEDIATE RISK 10-3 HIGH RISK 10-2
SLOWER KINETICS OF CLEARANCE IN T-ALL COMPARED TO PRE-B -ALL
FUTURE STUDIES
MICROARRAYS
THIS STUDY WAS FUNDED BY THE NCI GRANT FRA No N427-645
AND THE DEPARTMENT OF SCIENCE AND TECHNOLOGY, GOVT OF INDIA
THANKS TO
Dr T RAJKUMAR, SCIENTIFIC DIRECTOR
MR SUDHAKAR, SRF IN THE DEPT
DR RAJALEKSHMY, HEMATOPATHOLOGIST
MISS MEENA , GRADUATE TECHNICIAN
DR T G SAGAR ,DR ANITHA & DR S G RAMANAN
DR V SHANTA, CHAIRMAN , CANCER INSTITUTE(WIA)